Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The int-3 gene was originally identified based on its oncogenic effects in the mouse mammary gland. int-3 is a frequent target for insertional activation by MMTV proviral DNA in MMTV-induced mammary gland tumors (Gallahan and Callahan, 1987; Robbins et al., 1992; Sarkar et al., 1994). Tumor specific transcripts derived from the int-3 gene encode a protein homologous to the intracellular part of the Notch family of cell surface receptors. Exogenous expression of the int-3 oncoprotein has been shown to affect the growth and development of mammary epithelial cells. Over expression of the int-3 oncoprotein in a mouse mammary epithelial cells (HC11) promotes anchorage independent growth (Robbins et al., 1992). Expression of int-3 as an MMTV-LTR driven transgene in the mouse mammary gland results in abnormal development of the mammary gland and rapid development of undifferentiated mammary carcinomas (Jhappan et al., 1992). In the normal mouse mammary gland, endogenous int-3 protein has been detected in mammary stroma and epithelium (Smith et al., 1995).
Members of the Notch/lin-12 gene family were first identified in Drosophila and Caenorhabditis elegans through genetic analysis of mutations that alter cell fate decisions (for review see Artavanis-Tsakonas et al., 1995; Artavanis-Tsakonas and Simpson, 1991; Greenwald and Rubin, 1992). Drosophila Notch regulates multiple cell fate decisions that involve cell-cell interactions during fly development, for instance, control of cell fate decisions involving neural/epidermal specification in proneural clusters (Artavanis-Tsakonas and Simpson, 1991). The C. elegans lin-12 and glp-1 proteins are structurally related to Notch, and are also involved in cell fate specifications during development in the nematode (Greenwald, 1985; Yochem and Greenwald, 1989). Genetic analysis of Notch/lin-12 genes suggest that this family of genes controls binary cell fate decisions and inductive signaling that depend on cell-cell interactions (reviewed in Artavanis-Tsakonas et al., 1995; Greenwald, 1994; Greenwald and Rubin, 1992). Alternatively, Notch/lin-12 genes have been proposed to block cell differentiation, thus maintaining the competence of cells for subsequent cell-fate determination (Coffman et al., 1993; Fortini et al., 1993).
Notch/lin-12 genes encode transmembrane receptor proteins characterized by highly repeated, conserved domains. The amino terminus of Notch proteins encodes the extracellular domain and contains as many as 36 repeats of an EGF-like motif involved in ligand binding (Rebay et al., 1993), and three tandem copies of a Notch/lin-12 sequence motif of unknown function. The intracellular portion of Notch proteins is characterized by six tandem copies of a cdc10/ankyrin motif, thought to be a protein-protein interaction domain (Michaely and Bennett, 1992), and a PEST sequence motif which may represent a protein degradation signal (Rogers et al., 1986). In several systems, truncated forms of Notch/lin-12 proteins that contain an intact intracellular domain without most of the extracellular domain behave as constitutively activated receptors (reviewed in Artavanis-Tsakonas et al., 1995; Greenwald, 1994). The human Notch 1 orthologue, TAN-1, was first identified in independently isolated translocation breakpoints in acute T lymphoblastic leukemia and is predicted to encode a truncated product that has an intact intracellular domain but lacks most of the extracellular domain (Ellisen et al., 1991). Similarly, the int-3 oncoprotein encodes the intracellular domain of a Notch-like protein and thus has been proposed to act as an activated Notch receptor (Robbins et al., 1992).
Based on sequence similarity to Drosophila Notch, additional Notch-related genes have been isolated from mammals; including mouse (Franco Del Amo et al., 1993; Lardelli et al., 1994; Lardelli and Lendahl, 1993; Reaume et al., 1992), rat (Weinmaster et al., 1992; Weinmaster et al., 1991), and human (Ellisen et al., 1991; Stifani et al., 1992; Sugaya et al., 1994). To date, three Notch homologues, Notch1, Notch2, and Notch3, have been identified in the mouse and their embryonic expression patterns display partially overlapping but distinct patterns of expression that are consistent with a potential role in the formation of the mesoderm, somites, and nervous system (Williams et al., 1995). Abundant expression of Notch1, Notch2, and Notch3 is found in proliferating neuroepithelium during central nervous system development. Targeted disruption of the Notch1 gene in mice results in embryonic death during the second half of gestation (Conlon et al., 1995; Swiatek et al., 1994) and homozygous mutant embryos display delayed somitogenesis as well as widespread cell death, preferentially in neuroepithelium and neurogenic neural crest (Conlon et al., 1995; Swiatek et al., 1994).
The gene products of Drosophila Delta (Vassin et al., 1987) and Serrate (Fleming et al., 1990), and C. elegans Lag-2 (Henderson et al., 1994; Tax et al., 1994) and Apx-1 (Mello et al., 1994) are thought to act as ligands for Notch proteins. In the mouse, the orthologue of Delta, referred to a Dll1 (Delta-like gene 1), is expressed during embryonic development in the paraxial mesoderm and nervous system in a pattern similar to that of mouse Notch1 (Bettenhausen et al., 1995). A murine Serrate-related gene named Jagged has been identified and is partially co-expressed with murine Notch genes in the developing spinal cord (Lindsell et al., 1995).
The identification and expression analysis of a fourth murine Notch homologue is reported here. The fourth murine Notch homologue has been named Notch4 and the int-3 nomenclature has been reserved for the truncated oncogene. Although the intracellular domain of the int-3 oncoprotein shares homology with the Notch/Lin-12 protein family, a comparison of the full length Notch4 protein to that of the int-3 oncoprotein is now provided. The activated int-3 protein encodes only the transmembrane and intracellular domain of the Notch4 protein. The predicted amino acid sequence of Notch4 contains the conserved features of all Notch proteins, however Notch4 has 7 fewer EGF-like repeats compared to Notch1 and Notch2 and contains a significantly shorter intracellular domain. Notch4 is expressed primarily in embryonic endothelium and in adult endothelium and male germ cells.
This invention uses an established cell line, Rat Brain Microvessel Endothelial cells (RBE4 cells), to test Notch4 activity and to demonstrate that activated Notch4 and Notch4 ligand stimulate angiogenesis in these cells. RBE4 cells grown on collagen coated plates in the absence of bFGF or with low concentrations of bFGF (1 ng/ml), display a cobblestone morphology. In the presence of high concentration of bFGF (5 ng/ml), RBE4 cells exhibit a spindle shape morphology. When RBE4 cells reach confluency, they growth arrest and cells retain their cobblestone morphology. Post-confluent RBE4 cell cultures grown in the presence of 5 ng/ml bFGF, develop multicellular aggregates from the cobblestone monolayer. These three-dimensional structures are sprouts that extend above the monolayer and some of these sprouts will organize into curvilinear and bifurcating structures that float in the cell culture medium. The RBE4 cell sprouts and three-dimensional structures contain high activity of several enzymatic markers that are specific for differentiated microvessels. Thus, bFGF, a known angiogenic agent, induces angiogenesis of RBE4 cells which upon treatment with high concentrations of bFGF develop structures resembling capillaries (based on their morphological appearance and based on their expression of differentiated endothelial cell markers).
The Notch1 and Jagged genes have been previously described to be expressed in mouse endothelial cells.
Contrary to previous studies suggesting that Jagged and Notch inhibit angiogenesis (Zimrin et al. J Biol Chem. 1996), this invention demonstrates that activated Notch4 protein and Jagged protein contain a biological activity on RBE4 cells that is similar to the known angiogenic agent basic fibroblast growth factor (bFGF), suggesting that the above-described biological activity is an angiogenic activity.
This invention demonstrates that RBE4 cells express an endogenous Notch, since RBE4 cells that are programmed to express Jagged (a mammalian Notch ligand) have a similar phenotype when compared to RBE4 cells that express a constitutive activated Notch (int-3). This is consistent with previously published data showing that Notch4 is expressed in endothelial cells in vivo.
This invention demonstrates that RBE4 cells do not express sufficient levels of Notch4 ligand to activate the Notch4 receptor, since RBE4 cells programmed to express Notch4 do not exhibit a spindle form morphology when grown in the absence of bFGF.
This invention also demonstrates that over expression of Notch4 protein in RBE4 cells does not result in activation of the Notch4 receptor, because otherwise one would expect that these cells would have a similar phenotype to those cells that express a constitutive activated form of the Notch4 receptor.
This invention also demonstrates that bFGF mediated angiogenesis is unrelated to Notch4 mediated angiogenesis. This invention further suggests that Jagged or activated Notch4 induced activity can synergize or cooperate with bFGF.
Notch signaling is typically associated with cell fate decision. The finding that Notch activation stimulates capillary outgrowth suggests that Notch receptors must be activated to allow this process to occur. Therefore, Notch modulation provides a novel paradigm for regulating angiogenesis. Specifically, modulation of Notch signaling can be used to modulate angiogenesis either positively, by activating Notch signaling to stimulate angiogenesis or negatively, by blocking Notch signaling to block angiogenesis. This modulation would be distinct from previous proteins known to modulate angiogenesis. The induction or inhibition of angiogenesis in vivo can be used as a therapeutic means to treat a variety of diseases, including but not limited to cancer, diabetes, wound repair and arteriosclerosis.
This invention provides three major advantages over previously published paradigms. First, because Notch signaling is distinct from FGF signaling, this offers a different strategy to affect angiogenesis that may or may not be more effective then FGFs. Second, because Notch signaling establishes cell fate decision, by blocking Notch, angiogenesis may be blocked regardless of what other angiogenic factors are present. Third, Notch and FGF may be used synergistically to modulate angiogenesis.
This invention provides an isolated nucleic acid molecule, encoding a Notch4 protein. This invention provides a vector comprising the above-described nucleic acid. This invention also provides an isolated Notch4 protein.
This invention also provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to the DNA encoding Notch4 protein so as to permit expression thereof.
This invention also provides a mammalian cell comprising the above-described plasmid or vector.
This invention also provides a nucleic acid probe comprising a nucleic acid of at least 12 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid encoding Notch4 protein.
This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding Notch4 protein so as to prevent translation of the mRNA.
This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding activated Notch4 protein so as to prevent translation of the mRNA.
This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA encoding a Notch4 protein or an activated Notch4 protein.
This invention provides an antibody directed to a Notch4 protein. This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce activity of Notch4 protein by passing through a cell membrane and binding specifically with mRNA encoding Notch4 protein in the cell so as to prevent its translation and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
This invention provides a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of Notch4 protein and a pharmaceutically acceptable carrier.
This invention provides a pharmaceutical composition comprising an amount of an agonist effective to increase activity of Notch4 protein and a pharmaceutically acceptable carrier.
This invention provides the above-described pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to the Notch4 protein and a pharmaceutically acceptable carrier.
This invention also provides a method for determining whether a ligand can specifically bind to Notch4 protein comprising the steps of: a) contacting Notch4 protein with the ligand under conditions permitting formation of specific complexes between Notch4 protein and known Notch4 protein-binding ligands; b) determining whether complexes result from step (a), the presence of such complexes indicating that the ligand specifically binds to Notch4 protein.
This invention provides a method for determining whether a ligand can specifically bind to a Notch4 protein which comprises preparing a cell extract from cells transfected with and expressing DNA encoding the Notch4 protein, isolating a membrane fraction from the cell extract, contacting the membrane fraction with the ligand under conditions permitting binding of ligands to such Notch4 protein, detecting the presence of the ligand specifically bound to Notch4 protein, and thereby determining whether the ligand specifically binds to Notch4 protein.
This invention provides a method for determining whether a ligand is a Notch4 protein agonist which comprises contacting a cell transfected with and expressing nucleic acid encoding Notch4 protein with the ligand under conditions permitting activation of a functional Notch4 protein response by ligands known to be agonists of Notch4 protein, and detecting whether a functional increase in Notch4 protein activity occurs so as to determine whether the ligand is a Notch4 agonist.
This invention provides a method for determining whether a ligand is a Notch4 protein antagonist which comprises contacting a cell transfected with and expressing DNA encoding Notch4 protein with the ligand under conditions permitting the activation of a functional Notch4 protein, and detecting whether a functional decrease in Notch4 activity occurs so as to determine whether the ligand is a Notch4 protein antagonist.
This invention provides a method of treating an abnormality in a subject, wherein the abnormality is alleviated by the inhibition of Notch4 protein activity which comprises administering to a subject an amount of Notch4 antagonist effective to inhibit Notch4 protein activity.
This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by the activation of Notch4 activity which comprises administering to a subject an amount of a Notch4 agonist effective to promote Notch4 protein activity.
This invention provides a method of preparing isolated Notch4 protein which comprises: a) inserting nucleic acid encoding Notch4 protein in a suitable vector which comprises the regulatory elements necessary for expression of the nucleic acid operatively linked to the nucleic acid encoding Notch4 protein; b) inserting the resulting vector in a suitable host cell so as to obtain a cell which produces Notch4 protein; c) recovering the Notch4 protein produced by the resulting cell; and d) purifying the protein so recovered.
This invention also provides a method of modulating angiogenesis in a subject comprising administering to the subject an effective amount of agonist or antagonist of the Notch4 protein so as to promote or inhibit angiogenesis in the subject.
This invention also provides a method of promoting angiogenesis in a subject comprising administering to the subject an amount of Notch4 protein agonist effective to promote angiogenesis in the subject.
This invention also provides method of inhibiting angiogenesis in a subject comprising administering to the subject an amount of Notch4 protein antagonist effective to inhibit angiogenesis in the subject.
This invention also provides method of promoting angiogenesis comprising transducing selected cells, wherein the cells express activated Notch4 protein in an amount sufficient promote angiogenesis in the cells.
This invention provides a method of promoting angiogenesis comprising transducing selected cells which express Notch4 protein, wherein the cells express a Notch4 ligand in an amount sufficient to promote angiogenesis in the cells.
This invention provides a method of promoting angiogenesis comprising transducing selected cells, wherein the cells express a Notch4 protein and a Notch4 ligand in an amount sufficient to promote angiogenesis in the cells.
This invention provides a method of inhibiting angiogenesis comprising administering to cells expressing Notch4 protein an amount of a specific antibody effective to block binding of a ligand to Notch4 protein.
This invention provides a method of inhibiting angiogenesis comprising administering to cells expressing Notch4 protein an amount of a fragment of a specific antibody effective to block binding of a ligand to Notch4 protein.
This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by inhibiting angiogenesis comprising blocking Notch4 protein signaling in the subject.
This invention also provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by promoting angiogenesis comprising stimulating the Notch4 signaling pathway.